1. Field of the Invention
The invention relates to the peptides of the antigen Sm-D, which are recognized by antibodies in biological fluids, in particular of antibodies which are present in the body fluids of patients which suffer from systemic lupus erythematosus (SLE).
Furthermore, the invention relates to the use of these peptides and their sequential products for the diagnostics of SLE in vitro, as well as their use as a vaccine for the generation of tolerance mechanisms and for the preparation of immunogenic and antigenic compositions and of test kits.
In addition, the invention concerns antibodies which are induced in vivo by these peptides as well as a method for the determination of anti-Sm-D antibodies in biological fluids.
2. Brief Description of the Background of the Invention Including Prior Art
It is known that many autoantigens are positioned in a close spatial relationship. For example, the Sm and the various ribonucleoproteins RNP form a ribonucleoprotein complex, which is present in the core of eukariotic cells (snRNP). Anti Sm antibodies recognize various proteins of the snRNP complex, which are designated as B', B, D, E, F, and G. This complex probably plays a central role in the splicing of the pre-mRNA, wherein the Sm-D protein represents an important regulation protein, in that it is to regulate the binding of various ribonucleic acids.
It is ultimately undetermined which mechanisms lead to the formation of autoantibodies. It is noticeable that particularly important regulatory centers for the cell cycle are the goal of the autoantibodies of the SLE and of syndromes related to SLE. Upon the generation of the autoimmune diseases, particularly important regions are in general to be recognized by autoantigens and are to interfere with the physiological functioning of the particularly important regions of the autoantigens.
Bloom and coworkers could prove in connection with investigations of hybridoma cell lines, produced by means of hybridoma technique, that anti-Sm antibodies can also recognize deoxyribonucleic acid DNA (D. D. Bloom et al, Journal Immunol. 1993; 150 (4): 1579-1590). The authors concluded from this that the Sm-D protein by itself does not act as antigen for the formation of anti-Sm antibodies. Rather, it is assumed that both the Sm-D autoantigen and the deoxyribonucleic acid DNA, possibly as a complex, form the autoimmune agent. No binding capability of the Sm-D protein with the deoxyribonucleic acid DNA could be determined up to now. The physiological and pathophysiological importance of the Sm/deoxyribonucleic acid DNA complex is not known up to now.
Lebrun and coworkers carried out immunization tests at certain breeds of mice with deoxyribonucleic acid DNA, wherein they coupled the deoxyribonucleic acid DNA with a fusion protein, rich in arginine, and they could thereby generate a lupus nephritis (P. Lebrun et al, Lupus 1994; 3: 47-53). They also concluded that certain proteins, binding to the deoxyribonucleic acid DNA, are necessary in order to generate an autoimmune reaction.
In addition to the antibodies (Ad) against native or double-stranded deoxyribonucleic acid (dsDNA), anti-Sm antibodies are deemed to be a diagnostic marker for the systemic lupus erythematosus (SLE). In addition, a pathogenic role is attributed to the anti-Sm antibodies in the generation of damages to organs. The proof of the anti-Sm antibodies succeeds in Europe, contrary to the anti-ds-DNA antibodies, only in case of relatively few patients, whereas in the United States it can be determined in one third of the patients with SLE. The cause of this is considered to be a different ethnic composition of the population (N. Abuaf et al. Eur. J. Clin. Invest. 1990; 20: 354-359).
There are several methods of determining the presence of anti-Sm antibodies.
The immuno diffusion and countercurrent electrophoresis are relatively specific methods for a detection of anti-Sm antibodies, however, these methods are relatively insensitive and, in addition, time-consuming and expensive.
Anti-Sm antibodies can be detected by way of immuno blotting depending on the used antigen substrate in about 30% of the SLE patients also in Europe (G. Riemekasten and F. Hiepe, Zeitschrift arztlicher Fortbildung, 1992; 86: 217-222). It is possible based on this method to characterize the anti-Sm antibodies in a better way. Anti-Sm antibody-positive patients react amongst others with the B'B proteins as well as with the proteins D, E, F, and G of a small nucleic ribonucleoprotein complex (snRNP). While anti-B'B antibodies are not so specific for the SLE, the antibodies in particular against the D protein are deemed to be SLE specific.
This immuno blotting method is however associated with the disadvantage that it requires a substantial time expenditure and is expensive. In addition, a lot of experience is necessary in the connection with the evaluation.
Therefore, commercially available ELISA techniques are employed in many laboratories for detecting the presence of Sm antibodies, which ELISA techniques are characterized by a high sensitivity and by a relatively simple production practicability at a high sample throughput. These tests are for example performed on the basis of Sm purified from thymus extracts as a solid-phase antigen (printed patent document WO 90/10229) and of recombinant produced Sm antigen (L. A. Rokeach et al., Clinical Immunology and Immunopathology 1992; 65(3): 315-324). Rokeach and coworkers could isolate for the first time the cDNA of the Sm-D protein and could show that it codes a protein consisting of 119 amino acids.
Thus, the foundations were created for the generation of the ELISAs on the basis of synthesized peptides, cf. European printed patent document EP 0,295,719, wherein the application of cloned Sm-D was described for the diagnostics of the SLE.
A multitude of these, in part also commercially available tests are however relatively unstable, time-consuming and, based on false positive results due to cross reactions, less specific (printed patent document WO 90/10229).
In order to determine the main epitopes of the Sm-D protein, James and coworkers have subdivided the complete SmD protein into overlapping octapeptides and have investigated the reactivities in the ELISA relative to 15 SLE sera (James et al., Clin. exp. Immunol. 1994; 98: 419-426). Five epitopes were found, the main epitopes were also at the C-terminal end as well as epitopes of the amino acids 37-53, of the amino acids 69-76, and of the amino acids 5-12. Barakat and coworkers found three regions of the SmD protein (first region amino acids 1 through 20, second region amino acids 44 through 67, and third region amino acids 97 through 119) with which anti-SmD antibody-positive sera reacted which had been previously detected in immuno blotting (Barakat et al., Clin. exp. Immunol. 1990; 81: 256-262). 67% of the overall 18 anti-SmD antibody-positive sera reacted with the peptide 1-20, 89% with the peptide 44-67, and only 33% with the peptide 97-119. 165 of the SLE sera were tested relative to their reactivity in the ELISA with the peptides 1-20, 44-67, and 97-119, wherein positive reactions occurred in 59%, 37%, and 1%. Sabbatini and coworkers found positive reactions against the region 95-119 in 25% of the 48 SLE sera in the ELISA (A. Sabbatini et al., J. Rheumatol. 1993; 20: 1679-1683).
Short-chain peptides were used in connection with all these investigations and the number of the positive results of the SLE sera varied for the C-terminal end between 1% and 33% in the ELISA. Thus, these synthetically produced peptides are not associated with a substantial advantage relative to the commercially available tests. Only linear epitopes can be captured with this epitope mapping. The reactivity in connection with autoimmune diseases, however, is directed against intact autoantigens (E. M. Tan, J. Exp. Med. 1994; 179: 1083-1086).
In connection with a possible use of the complete SM-D antigen, there exists the danger that important epitopes are no longer accessible to the antibodies based on the secondary and tertiary structure. In addition, a new conformation can be generated by binding of parts of the Sm-D antigen to other autoantigens, preferably DNA.
A proof of a reactivity of the anti-La antibody, which is directed against a conformation epitope, could be furnished by way of peptide chains of the La protein of different lengths as a solid-phase antigen in the ELISA by Rischmueller and coworkers (M. Rischmueller et al., Clin. exp. Immunol. 1995; 101: 39-44).
The peptides of the Sm-D antigen and their use for the diagnostics of the SLE are described in the printed patent document WO 91/18920. These known peptides, wherein the amino acids are mainly directly N terminal, exhibit however only a low frequency of positive reactions (approximately 30% positive reactions).